Phosphatidylinositol 4,5-bisphosphate hydrolysis in turkey erythrocytes is regulated by P2y purinoceptors

CP Berrie, PT Hawkins, LR Stephens, TK Harden and CP Downes

Department of Cellular Pharmacology, Smith Kline and French Research Limited, Welwyn, England.

When intact [3H]inositol-loaded turkey erythrocytes were stimulated with the purinergic agonist ADP, there was a rapid increase (2.5-fold after 30 sec) in the intracellular content of [3H]inositol 1,4,5- trisphosphate, followed by increases in the levels of [3H]inositol bisphosphate and [3H]inositol 1,3,4,5-tetrakisphosphate (4-fold and 5- fold, respectively, after 3 min). [3H]inositol monophosphate levels did not rise in the first 3 min of ADP stimulation but increased slowly thereafter, demonstrating that the primary response of turkey erythrocytes to purinergic stimulation is hydrolysis of phosphatidylinositol 4,5-bisphosphate. Inositol phosphate accumulation was evoked by a P2y purinoceptor, as indicated by the rank order of potencies of a variety of purinergic agonists. 2-Methylthioadenosine 5'- triphosphate was the most potent agonist tested, with an EC50 value of 0.36 microM. High performance liquid chromatography analysis demonstrated the presence of three distinct inositol tetrakisphosphate isomers in [3H]inositol-loaded turkey erythrocytes, inositol 1,3,4,6- tetrakisphosphate [Ins(1,3,4,5)P4], inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and inositol 3,4,5,6-tetrakisphosphate. Prolonged stimulation with adenosine 5'-O-(2-thiodiphosphate), a nonhydrolyzable analogue of ADP, resulted in a 60-fold increase in the level of [3H]Ins(1,3,4,5)P4, whereas a substantial rise in the [3H]Ins(1,3,4,6)P4 fraction was also seen. These results indicate that turkey erythrocytes represent a valuable model system for studies of purinoceptor function as well as fundamental aspects of cell surface receptor-regulated phosphoinositide metabolism.

Volume 35, Issue 4, pp. 526-532, 04/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics

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